Rett Syndrome (RTT) is a devastating neurodevelopmental disorder and the leading known genetic cause of autism in girls. Mutations in the X-linked gene MECP2 (methyl-CpG binding protein 2) account for the vast majority of RTT cases. The neurobiology of MECP2 is fundamental to understanding the mechanisms of RTT and identifying therapeutics for the disorder. MeCP2 is an epigenetic modulator of gene expression that has recently been shown to interact significantly with microRNA machinery; these interactions are at the core of MeCP2 mechanisms. Multiple lines of evidence point to a role for MeCP2 in successive stages of brain development, including prenatal neurogenesis, postnatal development of connections and function, and experience-dependent synaptic plasticity. We hypothesize that the pleiotropic effects of MeCP2 are mediated in prenatal development via a set of early regulated miRNAs that influence neurogenesis; during postnatal development through a different set of miRNAs that regulate Insulin-like growth factor 1 (IGF1) signaling; and in late development into adulthood via a third set of miRNAs that influence synaptic function and plasticity. The goal of this proposal is to employ cutting-edge miRNA methodologies, in combination with stem cell, behavioral, two-photon imaging, and targeted electrophysiological approaches, to reveal the function of MeCP2-related miRNAs at different developmental stages. In aim 1, we will examine the role of MeCP2 and downstream miRNA-mediated pathways in prenatal neurogenesis, using isogenic human RTT model cell lines (aim 1a), 3-D cerebral organoids (aim 1b), and mouse models (aim 1c). Our findings to date implicate miR-199 and -214 in the aberrant regulation of prenatal neurogenesis as a result of MeCP2 deficiency; we will analyze the functional mechanisms and molecular pathways downstream of these miRNAs. In aim 2, we will determine the influence of postnatal MeCP2-regulated miRNAs on IGF1 signaling, and their potential role in RTT therapeutics. We will examine the regulation of LIN28a and the let-7 family of miRNAs downstream of BDNF, and their ability to regulate IGF1 expression, in Mecp2 deficient mice (aim 2a). We will investigate whether normalizing the levels of molecular alterations using the 2 adrenergic receptor agonist clenbuterol can positively impact survival and a range of phenotypes in Mecp2 deficient mice (aim 2b), along with synergistic interactions between clenbuterol and IGF1 as a potent mechanism-based combination therapeutic for RTT (aim 2c). In aim 3, we will examine the role of MeCP2 and late-expressed miRNAs such as miR-132 in regulating experience-dependent cortical plasticity. We will determine whether restoring expression of miR-132 in the visual cortex of Mecp2 mutant mice can restore normal age-dependent maturation of ocular dominance plasticity (aim 3a). We will also examine whether IGF1 (and subsequently, clenbuterol and the combination of clenbuterol and IGF1) upregulates miR-132 expression, and can act through its downstream mechanisms to influence plasticity (aim 3b).